Specific binding of o-phenanthroline at a DNA structural lesion.

DNA intercalators are found to recognize a DNA lesion as a high affinity receptor site. This lesion-specific binding is observed when one strand of a DNA double helix contains an extra, unpaired nucleotide. Our assay for binding controls for the effects of sequence with a series of oligodeoxynucleotide duplexes which are identical except for the location of the lesion, an extra cytidine. Scission of the series of oligodeoxynucleotides by the cuprous complex of ortho-phenanthroline (OP-Cu) indicates that OP-Cu binds at the lesion-specific stable intercalation site, suggesting that OP-Cu intercalates into DNA. The dispersion of OP-Cu scission sites over three residues is consistent with scission via a diffusible intermediate. The location of the scission sites, directly on the 3' side of the lesion, is consistent with minor groove binding in B DNA.     

Keratan-sulphate-containing proteoglycans in human osteochondrophytic spurs of the femoral head

Newly synthesized and endogenous proteoglycan was isolated from human femoral head osteochondrophytic spurs. 35SO4-containing keratan sulphate was measured by its susceptibility to endo-beta-D-galactosidase (keratanase) and comprised 15-17% of the two subpopulations of a proteoglycan monomer fraction (D1) resolved by Sepharose CL-2B chromatography (Kav (I), 0.22; (II), 0.78). The size of the newly synthesized keratan sulphate in these fractions was large (Mr greater than 7,000). The hydroxylamine cleavage product of a proteoglycan aggregate fraction (A1) which eluted in the void volume of a Sepharose CL-2B column was immunoreactive with an anti-keratan sulphate monoclonal antibody, 5-D-4. Unlike the proteoglycan aggregate A1 fraction from bovine nasal cartilage, immunoreactivity against 5-D-4 was also found in chromatographic fractions retarded by Sepharose CL-2B. These results lend additional support to our assertion that the osteophyte extracellular matrix consists of hyaline cartilage-type proteoglycan. Stimulation of osteophyte proliferation may be useful as a repair mechanism for resurfacing denuded areas of osteoarthritic femoral heads.     

Depletion of brainstem epinephrine stores by alpha-methyldopa: possible relation to attenuated sympathetic outflow

The antihypertensive effect of alpha-methyldopa (MD) is believed to be critically dependent on its ability to deplete endogenous catecholamines or cause the synthesis of false neurotransmitters. We used liquid chromatography with electrochemical detection (LCEC) and negative chemical ionization gas chromatography-mass spectrometry (GC-MS) for quantitation of catecholamines and MD metabolites in rat. MD intraperitoneally (100 mg/kg q12 hr X 12 days), significantly increased alpha-methylnorepinephrine (MNE) in brain (1.02 +/- 0.33 micrograms/g), heart (1.67 +/- 0.57 micrograms/g) and adrenal glands (114.93 +/- 50.47 micrograms/g) Endogenous norepinephrine (NE), epinephrine (E) and dopamine (DA) were reduced. ME levels were 2.19 +/- 0.44 micrograms/g (n = 6) in the adrenal gland but only 99 +/- 26 pg/g (n = 3) in the brainstem. MD-induced endogenous brainstem NE depletion was more than compensated by MNE production, but brainstem E depletion was not compensated for by a stoichiometric production of brainstem ME. We conclude (1) although ME is a metabolite of MD, it is present in extremely low concentrations in brainstem and (2) central epinephrine-containing neurons are depleted of neurotransmitter by MD therapy. If this selective epinephrine depletion occurs in the bulbospinal tract neurons responsible for maintaining sympathetic tone, then this effect could contribute to the antihypertensive effect of MD.     

Human renal clear cell carcinoma: Establishment and characterization of a new cell line (G-2101)

Summary A human cell line has been established from a renal adenocarcinoma rib metastasis of a 58-y-old male. This cell line has been maintained in continuous culture for 20 mo. through more than 50 passages. It displays simulataneous expression of the intermediate filaments cytokeratin and vimentin. Flow cytometric analysis of DNA content reveals a major hyperdiploid population. This work was supported in part by a grant from Triton Biosciences, Inc.     

Involvement of the chaperonin dnaK in the rapid degradation of a mutant protein in Escherichia coli.

The ability of Escherichia coli rapidly to degrade abnormal proteins is inhibited by mutations affecting any of several heat shock proteins (hsps). We therefore tested whether a short-lived mutant protein might become associated with hsps as part of its degradation. At 30 degrees C, the non-secreted mutant form of alkaline phosphatase, phoA61, is relatively stable, and very little phoA61 is found associated with the hsp dnaK. However, raising the temperature to 37 degrees C or 41 degrees C stimulated the degradation of this protein, and up to 30% of cellular phoA61 became associated with dnaK, as shown by immunoprecipitation and Western blot analysis. Also found in complexes with phoA61 were the hsps, protease La and grpE (but no groEL, or groES). The rapid degradation of phoA61 at 37 degrees C and 41 degrees C is in part by protease La, since it decreased by 50% in lon mutants. This process also requires dnaK, since deletion of this gene prevented phoA61 degradation almost completely (unless a wild-type dnaK gene was introduced). In contrast, the missense mutation, dnaK756, enhanced phoA61 degradation. The dnaK756 protein also was associated with phoA61, but this complex, unlike that containing wild-type dnaK could not be dissociated by ATP addition. Furthermore, in a grpE mutant, the degradation of phoA61 and the amount associated with dnaK increased, while in a dnaJ mutant, phoA61 degradation and its association with dnaK decreased. Thus, complex formation with dnaK appears essential for phoA61 degradation by protease La and some other cell proteases, and a failure of the dnaK to dissociate normally may accelerate proteolytic attack.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metastasis-associated alterations in phospholipids and fatty acids of human prostatic adenocarcinoma cell lines.

Metastatic variants of human prostatic adenocarcinoma cell lines (DU-145, LNCaP, and ND-1) were studied by using soft agar colony forming efficiency, nude mice tumorigenicity, in vitro invasion assay, and type IV collagenase assay. The DU-145 and ND-1 cell line showed higher metastatic potential than LNCaP. Lipids from DU-145, ND-1, and LNCaP cells were extracted and analyzed by thin-layer chromatography and gas-liquid chromatography. The major lipids were phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, fatty acids, and cholesterol. The sphingomyelin level was significantly higher in highly metastatic cells (DU-145 and ND-1) compared with the lower metastatic variant (LNCaP). The increase in the synthetic pathway and decrease in degradation pathway of sphingomyelin in microsomal fractions was sufficient to account for the measured increase in sphingomyelin in DU-145 cells compared with LNCaP cells. The major fatty acids of these lipids were palmitic (16:0), stearic (18:0), oelic (18:1), and arachidonic acid (20:4). The arachidonic acid level was significantly decreased in DU-145 and ND-1 compared with LNCaP cells. Electron microscopic studies showed no significant changes in the morphology of DU-145, ND-1, and LNCaP cells. The results of these investigations demonstrate for the first time that sphingomyelin and arachidonic acid contents are different in high and low metastatic variants of human prostatic adenocarcinoma cell lines.     

A single-amino-acid substitution in polyomavirus VP1 correlates with plaque size and hemagglutination behavior.

The plaque size and hemagglutination characteristics of five cloned wild-type strains of polyomavirus were determined. The strains fell into two groups, those with large or small plaques, each with distinctive hemagglutination behavior at different temperatures and pHs. The nucleotide sequence of VP1, the major capsid protein of the virus, was determined for each of the viral strains. The PTA (large-plaque) and RA (small-plaque) strains differed only at residue 92 of VP1, where there is a glutamic acid or glycine, respectively (R. Freund, A. Calderone, C. J. Dawe, and T. L. Benjamin, J. Virol. 65:335-341, 1991). The same amino acid difference in VP1 correlated with plaque size and hemagglutination properties of the other sequenced viruses. Mutagenesis converting amino acid 92 from glutamic acid to glycine converted the plaque size and hemagglutination behavior of the large-plaque PTA strain to that of a small-plaque strain. Furthermore, PTA and RA VP1 proteins produced in Escherichia coli behaved as their parental viruses did in hemagglutination assays. These results demonstrate that amino acid residue 92 of VP1 is involved in determining the plaque size and hemagglutination behavior of polyomavirus and strongly suggest that this region of the VP1 polypeptide interacts directly with cell receptors.     

Integumental lesions caused by ectoparasites in a wild population of the side-blotched lizard (Uta stansburiana)

Histopathological effects of ectoparasites on integument were examined for a wild population of the side-blotched lizard Uta stansburiana. These included the trombiculid Neotrombicula californica, the pterygosomatid mite Geckobiella texana; the macronyssid mite Ophionyssus natricis (Macronyssidae) and the ixodid tick Ixodes pacificus. A diffuse inflammatory response occurred at the site of chigger and tick attachment which consisted of histiocyte, heterophil, fibroblast and lymphocyte infiltration that often extended into the dermis. Granuloma formation also was noted. The most prevalent parasite was N. californica which frequently occurred in large aggregations above the eyelids. Ectoparasites were most abundant from February through April.